Background: The aim of this study was to evaluate the difference between pre- and post-operative radiotherapy on the progress of ossification after free fibula flap reconstruction of the mandible using three-dimensional (3D) analysis. Methods: A total of 38 free fibula reconstructions of the mandible were evaluated retrospectively for ossification between bone segments by measuring Hounsfield Units (HU) in at least two postoperative computer tomography scans (average of 2.4 scans per patient; around the 5th, 12th, 16th, and 19th month postoperative). Three subgroups were created according to the time of irradiation: preoperative radiotherapy (preORT) (n = 11), postoperative radiotherapy (postORT) (n = 16), and patients without any radiation therapy (n = 11) as the control group (noRT). HU in eight regions of interest (ROI) and overlapping surfaces between segments per contact point, as well as influencing factors, were analyzed. Results: The fastest progress in gain of HU ossification with a difference of 0.30 HU/day was observed in noRT compared to preORT (p = 0.002). postORT was −0.24 HU/day slower than preORT (p = 0.005). Original and grafted bone showed a significantly slower HU uptake than between two graft segments with −84.18 HU/day (p < 0.001). Moreover, a larger initial overlapping surface between the segments in cm2 resulted in a higher rise of HU/day (p < 0.001). Conclusions: 3D analysis of post-reconstructive CT scans shows prolonged ossification of mandible reconstructions by free fibula after head and neck radiation. The effect is distinct in cases with post-operative adjuvant radiotherapy. The effects of radiotherapy on ossification may be minimized by a larger initial contact surface and improved operational techniques. Moreover, HU longitudinal measurements and 3D analysis offer new perspectives for clinical evaluation of successful bony healing.